Hodgkin's disease is a malignant lymphoma of mixed cell type which is usually classified according to the Lukes-Butler classification [Lukes et al., Cancer Res. 26, 1311 (1966)]. Cases of Hodgkin's disease of all four major histologic subtypes (lymphocyte predominance, mixed cellularity, lymphocyte depleted, and nodular sclerosis) contain variable numbers of cells that characterize the Hodgkin's disease process. The largest and most characteristic of these cells are termed Reed-Sternberg cells. The cell is typically binucleate, multinucleate, or has a polylobulated nucleus. Mononuclear variants of this cell are termed Hodgkin's cells. Morphological and immunohistochemical studies suggest that lymphocyte predominant Hodgkin's disease is a B-cell neoplasm and as such is distinct from other subtypes of Hodgkin's disease [see e.g. Pinkus, G. S. and Said, J. W., Am. J. Pathol. 133, 211-217 (1988); Burns et al., Am. J. Surg. Pathol. 8, 253-261 (1984); Hansmann et al., J. Cancer Res. Clin. Oncol. 114, 405-410 (1988)]. The cellular derivation of other types of Hodgkin's disease remains controversial.
The non-Hodgkin lymphomas are traditionally classified by standard histologic methods. However, since this classification provides little information about the origin or biology of these tumors, recently immunologic classification, emphasizing the principal immunologic cell type in the lymphoma, is favored. B cell-type lymphomas are at present classified according to the microscopic features of the cells, especially the nuclei. These are thought to represent different stages of differentiation of B cells between stem cells and plasma cells [see "Basic & Clinical Immunology" 3rd Edition, Fundenberg, H. H., Stites, D. P., Caldwell, J. L., Wells, J. V., Eds., Los Altos, Calif., 1980, Chapter 31]. The summary of an NCI-sponsored study of classification of non-Hodgkin's lypmomas was published in Cancer 49, 2112-2135 (1982).
The development of Hodgkin's and various B cell lines has permitted the production of monoclonal antibodies against such cell lines, and the identification of antigen molecules recognized by the antibodies.
The Fourth International Workshop of Leukocyte Differentiation Antigens (1989) listed no less than 78 major cluster designations (CDs1-78) for leukocyte antigens, with several CDs including more than one related antigens (e.g. CD11A, CD11B, CD11C, etc.). The molecular weights of the majority of these antigens have been defined. Of the 78 different CD antigens, approximately one dozen show some specificity for B cells (e.g. CDs 19-24, CD32, CD37, CD40, CDs 73-77). Only a minority of these show restricted specificity for B cells, and most are expressed on a limited number of other cell types.
Antigens that show some preferential expression on Reed-Sternberg cells and their mononuclear variants, Hodgkin's cells, in Hodgkin's disease include CD15, CD30, and CD74. CD15 was detected by antibody LeuM1, CD30 by antibody Ki-1 and related antibodies, and CD74 by antibody LN2. LeuM1 is, for example, described in the following publications: Hsu and Jaffe, Am. J. Clin. Pathol. 82, 29-32 (1984); Pinkus et al., Am. J. Pathol. 119, 244-252 (1985); and Hsu et al., JNCI 77, 363-370 (1986). Ki-1 and related antibodies are disclosed in numerous scientific publications, including Schwab et al., Nature 299, 65-67 (1982); Stein et al., Int. J. Cancer. 30, 445-449 (1982); O'Connor et al., Histopathology 11, 733-740 (1987). Articles concerning antibody LN2 include Epstein et al., J. Immunol. 133, 1028-1036 (1984) and Sherrod et al., Cancer 57, 2135-2139 (1986).
A further anti-Reed-Sternberg cell antibody, HeFi-1 is disclosed by Hecht et al., J. Immunol. 134. 4231-4236 (1985).
All of these antibodies identify only a portion of Reed-Sternberg cells in some of the subsets of Hodgkin's disease, and their specificity is not satisfactory. For example, upon more extensive study, the reactivity of Ki-1 appears not to be restricted to Reed-Sternberg cells, but includes the malignant cells of some of the non-Hodgkin's lymphomas, as well as a subset of apparently normal cells, the identity of which is not yet known, but which show some features of myeloid cells [Stein et al., Int. J. Cancer 29, 283-290 (1982)].
Monoclonal antibodies EPB-1 and EPB-2, reactive with human lymphoma, are described by Pawlak-Byczkowska et al., Cancer Research 49, 4568-4577 (1989). EPB-1 is reported to be specific to normal and malignant B cells and to Hodgkin's disease related cells, and is identified as having IgG.sub.1 isotype. Its antigen has an estimated molecular weight of 35,000. The immunogen agent used to make EPB-1 was a B cell lymphoma.
Functional aspects of the antigen molecules recognized by any of these antibodies have not yet been reported. In addition, although there have been some sporadic reports describing the existence of inhibitory or cytotoxic factors in Hodgkin's disease [Taylor, C. R., "Hodgkin's Disease and the Lymphomas", Annual Research Reviews, D. Horrobin, Series ed. Churchill Livingston/Eden Press, London/New York, 1980], almost no information is available with respect to mechanisms of regulation of growth and differentiation of Hodgkin's or Reed-Sternberg cells, other than the general .concept that the mononuclear Hodgkin's cell is the proliferating element from which the Reed-Sternberg cells are derived [Taylor, C. R., "Upon the enigma of Hodgkin's disease and the Reed-Stenberg cell." In: Controversies in the Management of Lymphomas II., J. M. Bennet, ed. Martinus Nijhoff Publishers, Boston, pp. 91-110, 1983; Taylor, C. R., Recent Results Cancer Res., 64, 214-231 (1978)].
Immunophenotypic characterization of lymphomas by monoclonal antibodies has proved a valuable adjunct to histologic diagnosis and has facilitated understanding of the lineage of certain lymphomas.
Monoclonal antibodies detecting various antigens have been used or proposed for a number of purposes in research, and for diagnostic studies of leukemias and lymphomas in men and animals. The techniques employed include:
1. Leukocyte identification by phenotype, utilizing flow cytometry, immunofluorescence, immunoenzyme techniques, or immuno electron microscopy. PA1 2. Leukocyte separation techniques, including flow cytometry and panning. PA1 3. Identification and classification of leukemias. PA1 4. Radioimmunimaging of lymphomas in animals and man. PA1 5. Radioimmunotherapy of lymphomas in animals and man. PA1 6. Studies of leukocyte differentiation, maturation and function in experimental models and human disease. PA1 a molecular weight of about 36,000 Dalton; PA1 the presence of an epitope recognized by antibody to the protein; PA1 specific expression by Hodgkin's cells and Reed-Sternberg cells in all subsets of Hodgkin's disease, and by activated and early proliferating B cells; PA1 no expression by T cells; PA1 capability of reacting with its antibody in both frozen and fixed/paraffin embedded tissues; PA1 a function associated with the growth of cells capable of the expression of this antigen protein. PA1 IgG.sub.3 subtype; PA1 reactivity with Hodgkin's cells and Reed-Sternberg cells in all subsets of Hodgkin's disease, and with activated and early proliferating B cells; PA1 lack of reactivity with T cells; PA1 capability of reacting with the corresponding antigen in both frozen and fixed/paraffin embedded tissues; PA1 reversible, dose-dependent growth-inhibitory effect on cells capable of expressing the corresponding antigen. PA1 disrupting the membranes of said cells; PA1 preparing cell extract containing solubilized proteins; PA1 contacting said extract with a monoclonal antibody specific to said protein; PA1 separating the fraction containing protein reacting with said monoclonal antibody; and PA1 isolating said protein. PA1 obtaining monoclonal antibody that is specific to the antigen protein, PA1 contacting said antibody with tissue or cells obtained from a mammal to be diagnosed; and PA1 detecting the antigen protein, if present, as well as assay kit for performing such method. PA1 obtaining monoclonal antibody specific to an antigen having the above properties, said antibody being labeled; PA1 labeling said antibody; PA1 contacting said labeled antibody with tissue obtained from a mammal; and PA1 imaging said label.
However, the vast majority of the antibodies described to date recognize epitopes that are sensitive to the process of fixation or embedment in paraffin wax. Such antibodies detect their corresponding antigens only in frozen sections exposed to minimal fixation (such as 10 seconds in acetone), and do not detect the corresponding antigens following formalin or B5 fixation and embedment in paraffin. This fact hampers the utilization of many of these antibodies for diagnostic purposes in which only fixed and paraffin embedded sections are available. Thus, particular diagnostic importance is attached to those antibodies that reliably detect their corresponding antigens in fixed tissues. Since this fact has been realized, the number of such antibodies available has been increased, but still constitutes only a small minority overall.
Accordingly, for successful diagnosis of lymphomas, antibodies that are sufficiently specific to the lymphoma to be identified, and detect the corresponding antigens following fixation and embedment in paraffin, are required.
With regard to the identification of Hodgkin's disease, of the above-mentioned antibodies LeuM1 and LN2 are both reactive in paraffin embedded tissues. Antibodies related to Ki-1 which are effective in paraffin embedded tissues, also have become available. However, as hereinabove mentioned, all three of these antibodies identify only a portion of Reed-Sternberg cells in some of the subsets of Hodgkin's disease. None of them identify Reed-Sternberg cells in all cases of Hodgkin's disease. EPB-1 is also reported to remain active after fixation and paraffin embedment, and appears to have a better specificity to B cells and Hodgkin's disease related cells than any of the earlier published antibodies.
However, there is no disclosure of the functional aspects of the antigens identified by any of these antibodies on Reed-Sternberg and Hodgkin's cells. Whereas the literature contains many reports describing the existence of inhibitory or cytotoxic factors in the serum of patients with Hodgkin's disease, the corresponding antigens have not been identified and little information is available with respect to the mechanisms of regulation of growth and differentiation of Hodgkin's or Reed-Sternberg cells.
There is a great need for monoclonal antibodies with specificity and high reactivity to Reed-Sternberg cells, which retain their immunoreactivity in tissues that have been fixed and embedded in paraffin. Such antibodies would have wide applications, since paraffin sections remain the standard in diagnostic histopathology, based upon convenience, safety, superior morphology preservation, and economic factors. Additionally, it would be desirable to identify antigen(s) specifically expressed on Hodgkin's disease related cells, which have regulatory functions in the growth and/or differentiation of such cells. Study of the function of such antigens would be an invaluable tool in understanding and ultimately, treating Hodgkin's disease.